Antibiotic LL-D42067β

ABSTRACT

Antibiotic LL-D42067β derived by aerobic fermentation of the microorganism Actinomadura madurae subspecies simaoensis NRRL 15734, useful as an antibacterial and antiparasitic agent.

This application is a division of application Ser. No. 593,159 filedMar. 26, 1984 which is now U.S. Pat. No. 4,578,468 (1986).

SUMMARY OF THE INVENTION

This invention relates to a new antibacterial and antiparasitic agentdesignated LL-D42067β, to its production by fermentation, to methods forits recovery and concentration from crude solutions and to processes forits purification. The present invention includes within its scope thebiologically pure culture which produces the antibiotic.

The structure and relative stereochemistry of LL-D42067β have beenelucidated and is shown below. ##STR1##

The pysico-chemical characteristics of LL-D42067β are described below:

(1) Molecular weight: 521 (FAB-MS)

(2) Molecular formula: C₂₇ H₂₃ NO₁₀

(3) Specific optical rotation: [α]_(D) ²⁶ =+770±10° (C 0.165, DMF);

(4) Ultraviolet absorption spectra: as shown in FIG. I

    ______________________________________                                        UV.sub.MAX.sup.CH.sbsp.3.sup.OH =                                                              212 nm (ε 28,200)                                                     253 nm (ε 33,200)                                                     318 nm (ε 13,200)                                                     378 nm (ε 19,700)                                                     393 nm (ε 21,700)                                    UV.sub.MAX.sup.0.1N HCl =                                                                      211 nm (ε 13,900)                                                     253 nm (ε 18,200)                                                     318 nm (ε 7,140)                                                      372 nm (ε 9,950)                                                      388 nm (ε 10,500)                                    UV.sub.MAX.sup.0.1N NaOH =                                                                     216 nm (ε 31,200)                                                     251 nm (ε 11,700)                                    ______________________________________                                    

(5) Infrared absorption spectrum: as shown in FIG. II (KBr disc): 3400,1646, 1620, 1545, 1463, 1252, 1195, 1020 cm⁻¹ ;

(6) Proton nuclear magnetic resonance spectrum (CDCl₃): as shown in FIG.III, and described in Table I;

(7) Carbon-13 nuclear magnetic resonance spectrum (CD₃ OD/CDCl₃): asshown in FIG. IV and described in Table II.

                  TABLE I                                                         ______________________________________                                        Proton NMR Data for LL-D42067 β                                          δ*                                                                             No. of Hydrogen                                                                              Multiplicity**                                                                           J (H)                                        ______________________________________                                        1.88   2              m                                                       2.30   2              m                                                       2.38   3              s                                                       2.57   2              m                                                       3.69   1              d,d        4.5, 14.3                                    3.87   3              s                                                       4.79   1              m                                                       4.81   1              d,d        4.5, 12.7                                    5.06   1              m                                                       5.32   1              d          5.8                                          5.53   1              d          5.8                                          6.54   1              s                                                       10.1   1              s                                                       12.8   1              s                                                       12.9   1              s                                                       ______________________________________                                         *CDCl.sub.3, ppm downfield from TMS.                                          **s = singlet; d = doublet; m = multiplet.                               

                  TABLE II                                                        ______________________________________                                        Carbon-13 NMR Data for LL-D42067β                                        Carbon     Chemical Shift (ppm)*                                              ______________________________________                                        1          19.1                                                               2          25.9                                                               3          26.9                                                               4          29.7                                                               5          62.0                                                               6          62.2                                                               7          65.0                                                               8          72.8                                                               9          91.1                                                               10         100.1                                                              11         110.5                                                              12         110.6                                                              13         111.7                                                              14         114.7                                                              15         119.2                                                              16         127.2                                                              17         128.8                                                              18         136.1                                                              19         136.3                                                              20         137.0                                                              21         138.1                                                              22         148.9                                                              23         151.9                                                              24         153.0                                                              25         165.3                                                              26         167.2                                                              27         183.6                                                              ______________________________________                                         *CD.sub.3 OD/CDCl.sub.3, ppm downfield from TMS.                         

DETAILED DESCRIPTION OF THE INVENTION

The new antibacterial agent designated LL-D42067β is formed during thecultivation under controlled conditions of a new strain of a newsubspecies of Actinomadura madurae. This new strain is maintained in theculture collection of the Medical Research Division, American CyanamidCompany, Pearl River, N.Y. as culture number LL-D42067. A viable cultureof this new microorganism has been desposited with the Patent CultureCollection Laboratory, Northern Regional Research Center, U.S.Department of Agriculture, Peoria, Ill. 61604, and has been added to itspermanent collection. It is freely available to the public in thisdepository under its accession number NRRL 15734.

Culture LL-D42067 was isolated from a soil sample from San Simao,Brazil. The culture was taxonomically characterized and was identifiedas a new subspecies of Actinomadura madurae, designated Actinomaduramadurae subspecies simaoensis.

Observations were made of the cultural, physiological and morphologicalfeatures of the culture in accordance with the methods detailed byShirling and Gottlieb [Intern. J. System. Bacteriol., 16:313-340 (1966)]and Gordon, et al. [Intern. J. System. Bacteriol., 24:54-63 (1974)]. Thechemical composition of the cell walls of the culture was determinedusing the method of Lechevalier, et al. [Adv. Appl. Microbiol., 14:47-72(1971)]. Details are recorded in Tables III-V, and a general descriptionof the culture is given below. Underscored descriptive colors are takenfrom Kelly and Judd [Nat. Bur. Stand., Spec. Publ., 440 (1976)] and theaccompanying Intersociety Color Council, National Bureau of StandardsCentroid Color Charts.

Growth Characteristics

Table III describes the cultural characteristics of culture LL-D42067 onvarious agar media which were selected from those recommended by theInternational Streptomyces Project Committee (hereinafter referred to as"ISP").

Micromorphology

Microscopic examination of the strain showed it to form short chains ofconidia on aerial hyphae which were slightly hooked to short-spirals (upto three turns). The spore surfaces were smooth when observed byelectron microscopy, distinguishing this isolate from A. verrucosopora.

Cell Wall Composition

Whole cell analyses showed the strain to contain meso diaminopimelicacid (DAP) and the sugar 3-O-methyl-D-galactose (madurose); thus itfalls into whole cell pattern type B. The cell wall composition was ofthe type III (meso DAP, glutamic acid, alanine, muramic acid andglucosamine) and the phospholipid pattern of type PIV (phosphatidylethanolamine and/or methylethanolamine plus unknownglucosamine-containing phospholipids). These data support the assignmentof the strain to the genus Actinomadura. The PIV phospholipid type isnot typical for A. madurae, which is usually PI.

Physiological Reactions

The physiological reactions of strain LL-D42067 were examined using boththe ISP system, Shirling and Gottlieb [Intern. J. Syst. Bacteriol.,16:313-340 (1966)] and the Gordon tests, Gordon, et al. [Intern. J.Syst. Bacteriol., 24:54-63 (1974)]. The utilization pattern of thestrain on ISP carbohydrate media is given in Table IV, along with thoseof other members of the genus reacting similarly. Culture LL-D42067resembles the Actinomadura madurae and Actinomadura verrucosoporagroups. As indicated above, however, it differs from Actinomaduraverrucosopora in having smooth spore walls. A comparison of reactions inthe Gordon test series of Actinomadura madurae (Gordon's data; seereference above) and LL-D42067, summarized in Table V, revealeddifferences only in amylase production and acid from glycerol andraffinose. Since amylase production and raffinose utilization have beenfound to be variable in Actinomadura madurae [Goodfellow, N., et al., J.Gen. Microbiol., 112:95-111 (1979)], the glycerol reaction remains theonly physiological difference of LL-D42067 from this taxon.

Since strain LL-D42067 is the same as Actinomadura madurae in allproperties evaluated except for its glycerol reaction and its PIVphospholipid pattern, it has been assigned to the taxon Actinomaduramadurae as a subspecies designated Actinomadura madurae subspeciessimaoensis.

                  TABLE III                                                       ______________________________________                                        Cultural Characteristics of LL-D42067 Actinomadura                            madurae subspecies simaoensis on ISP Morphological Media                                   Aerial    Vegetative   Soluble                                   Agar Medium Mycelium   Mycelium     Pigment                                   ______________________________________                                        Yeast extract, Malt                                                                       White, sparse                                                                            Medium orange-                                                                             None                                      extract (ISP 2)        brown-I53*                                             Inorganic Salts                                                                           Colorless  Colorless    None                                      Starch (ISP 4)                                                                Glucose Asparagine                                                                        Colorless  Colorless    None                                      (ISP 5)                                                                       Oatmeal (ISP 3)                                                                           Sparse     Light orange-                                                                              None                                                  pinkish-white                                                                            brown-I52*                                             ______________________________________                                         *I = ISCC Color charts                                                   

                  TABLE IV                                                        ______________________________________                                        Comparison of Carbohydrate Utilization Reactions of                           LL-D42067 With Related Actinomadura spp.                                      Carbohydrate                                                                           LL-D42067  A. madurae.sup.(a)                                                                        A. verrucosopora.sup.(a)(b)                   ______________________________________                                         .sub.=L-arabinose                                                                     +          +          +                                               .sub.=D-fructose                                                                      +          +          +                                              I-inositol                                                                             -          variable   variable                                        .sub.=D-mannitol                                                                      +          +          +                                              raffinose                                                                              -          -          -                                              rhamnose +          +          +                                              sucrose  +          +          +                                               .sub.=D-xylose                                                                        +          +          +                                              ______________________________________                                         .sup.(a) Goodfellow, M., et al., J. Gen. Microbiol., 112:95-111 (1979).       .sup.(b) Nonomura, H. and O'Hara, Y., J. Ferm. Technol., 49:904-912           (1971).                                                                  

                  TABLE V                                                         ______________________________________                                        Gordon Test Reactions of LL-D42067                                                                    A. madurae                                                            LL-D42067                                                                             (Gordon Data*)                                        ______________________________________                                        Degradation/Transformation of                                                 Casein            +         +(98)                                             Xanthine          -         -                                                 Hypoxanthine      +         +(98)                                             Tyrosine          +         +(91)                                             Adenine           -         -                                                 Production of                                                                 Amylase           -         +                                                 Gelatinase        +         +                                                 Phosphatase       -         ND                                                Nitrate Reductase +         +(98)                                             Urease            -         -                                                 Esculinase        +         +(98)                                             Growth on/in                                                                  5% Sodium Chloride                                                                              -         ND                                                Salicylate        -         ND                                                Lysozyme Broth    -         -(91)                                             Utilization                                                                   Acetate           +         +                                                 Benzoate          -         -(94)                                             Citrate           -         +(83)                                             Lactate           +         ND                                                Malate            +         +(84)                                             Mucate            -         -                                                 Oxalate           -         ND                                                Propionate        -         ND                                                Pyruvate          +         ND                                                Succinate         +         +(83)                                             Tartrate          -         -                                                 Growth at                                                                     10° C.     -         -                                                 45° C.     +         -(66)                                             53° C.     -         -                                                 Acid from                                                                     Adonitol          +         +(91)                                             Arabinose         +         +                                                 Cellobiose        +         +                                                 Dextrin           +         ND                                                Dulcitol          -         -                                                 Erythritol        -         -                                                 Fructose          +         ND                                                Galactose         +         +(84)                                             Glucose           +         +                                                 Glycerol          -         +                                                 Inositol          -         +(60)                                             Lactose           -         +(55)                                             Maltose           -         +(53)                                             Mannitol          +         +                                                 Mannose           +         +(94)                                             Melibiose         -         -                                                 α-Methyl- .sub.=D-glucoside                                                               -         -                                                 Raffinose         variable  -                                                 Rhamnose          +         +                                                 Salicin           +         ND                                                Sorbitol          -         -                                                 Sucrose           +         ND                                                Trehalose         +         +(96)                                             Xylose            +         +                                                 β-Methyl- .sub.=D-xyloside                                                                 +         ND                                                ______________________________________                                         *Percentages of cultures showing reaction given in parentheses if not         100%?                                                                         ND = Not determined.                                                     

For the production of this new antibacterial and antiparasitic agent thepresent invention is not limited to this particular organism or toorganisms fully answering the above growth and microscopiccharacteristics, which are given for illustrative purposes only. Infact, it is desired and intended to include the use ofnaturally-occurring mutants of this organism as well as induced mutantsproduced from this organism by various mutagenic means known to thoseskilled in the art such as exposure to nitrogen mustard, X-rayradiation, ultraviolet radiation, N'-methyl-N'-nitro-N-nitrosoguanidine,actinophages and the like. It is also desired and intended to includeinter- and intraspecific genetic recombinants produced by genetictechniques known to those skilled in the art such as, for example,conjugation, transduction and genetic engineering techniques.

The in vitro antimicrobial spectrum of LL-D42067β was determined by theagar plate dilution method with Mueller-Hinton agar and an inoculum ofeach test organism of approximately 10⁴ colony forming units deliveredby the Steers replicating device. The minimal inhibitory concentration(MIC) in mcg/ml was defined as the lowest concentration of LL-D42067βthat inhibitied visible growth after 18 hours incubation at 35° C.

The results, summarized in Table VI, show that LL-D42067β was activeversus gram-positive bacteria and moderately active against yeasts.

                  TABLE VI                                                        ______________________________________                                        Antimicrobial Spectrum of LL-D420678β                                    Test Organism            MIC(mcg/ml)                                          ______________________________________                                        Candida albicans CA 300      512                                              Saccharomyces cerevisiae                                                                       Y 15        512                                              Mycobacterium smegmatis                                                                        ATCC 607    512                                              Bacillus subtilis                                                                              ATCC 6633   4                                                Bacillus cereus  LL No. 4    ≦0.06                                     Enterococcus     OSU 75-1    1                                                "                SM 77-15    2                                                Streptococcus                                                                           faecalis   ATCC 29212  1                                            "         mutans     ATCC 27352-1                                                                              0.25                                         "         "          BHI (b)     0.25                                         "         sanguis    G9B (a)     0.5                                          Staphylococcus epidermidis                                                                     CMC 83-56   0.5                                              "         "          ATCC 12228  0.25                                         "         aureus     Smith       0.5                                          "         "          LL No. 14   0.5                                          "         "          LL No. 27   ≦0.06                                 "         "          LL No. 45   0.12                                         "         "          ATCC 25923  0.25                                         Micrococcus luteus                                                                             PC 1001     ≦0.06                                     Escherichia coli No. 311     512                                              "                ATCC 25922  512                                              Acinetobacter calcoaceticus                                                                    STFD 79-17  512                                              ______________________________________                                    

The antibiotic LL-D42067β derives utility from its antibacterial andantiparasitic activities. For example, the antibiotic may be used in thesuppression of intestinal bacterial flora, as a topical antibacterialagent or antiseptic against gram-positive bacteria and as a generaldisinfactant for surfaces such as instruments. It may also be useful asan antiprotozoal agent in the treatment of malaria. In addition to itsantimicrobial and antiparasitic activity LL-D42067β is effective as ananticoccidial agent in poultry. This utility is the subject of acopending application for United States Letters Patent.

In therapeutic use, the compound of this invention may be administeredin the form of conventional pharmaceutical compositions appropriate forthe intended use. Such compositions may be formulated so as to besuitable for oral or topical administration. The active ingredient maybe combined in admixture with a nontoxic pharmaceutically acceptablecarrier, which carrier may take a wide variety of forms depending on theform of preparation desired for administration, i.e., oral or topical.

General Fermentation Conditions

Cultivation of Actinomadura madurae subspecies simaoensis NRRL 15734 maybe carried out in a wide variety of liquid culture media. Media whichare useful for the production of this novel antibiotic LL-D42067βinclude an assimilable source of carbon, such as dextrin, sucrose,molasses, glycerol, etc.; an assimilable source of nitrogen such asprotein, protein hydrolysate, polypeptides, amino acids, corn steepliquor, etc.; and inorganic anions and cations, such as potassium,sodium, ammonium, calcium, sulfate, carbonate, phosphate, chloride, etc.Trace elements such as boron, molybdenum, copper, etc., are supplied asimpurities of other constituents of the media. Aeration in tanks andbottles is supplied by forcing sterile air through or onto the surfaceof the fermenting medium. Further agitation in tanks is provided by amechanical impeller. An antifoam agent such as silicone oil may be addedas needed.

General Procedure for the Isolation of LL-D42067β

Antibiotic LL-D42067β is recovered from the whole harvest mash byfiltration through a medium such as diatomaeous earth, extraction into asolvent such as ethyl acetate, concentration to a syrup, partitioningbetween heptane and methanol and concentration of the methanol phase toa residue. This residue is triturated with hexane, then concentrated toa residue which is dissolved in a mixture of equal parts acetonitrileand water and then evaporated giving a precipitate. This precipitate ispurified by preparative reverse phase high performance liquidchromatography (HPLC) using the system acetonitrile:water:acetic acid(3000:6000:5). The active fractions are combined, evaporated to anaqueous suspension and extracted with ethyl acetate which is evaporatedto obtain the pure LL-D42067β.

EXAMPLE 1 Inoculum Preparation

A typical medium used to grow the various stages of inoculum wasprepared according to the following formula:

    ______________________________________                                        Dextrose         1.0%                                                         Dextrin          2.0%                                                         Yeast extract    0.5%                                                         NZ Amine A ®.sup.1                                                                         0.5%                                                         Calcium carbonate                                                                              0.1%                                                         Water qs         100%                                                         ______________________________________                                         [.sup.1 A pancreatic digest of casein, registered trademark of Sheffield      Chemical, Norwich, New York                                              

This medium was sterilized. A 100 ml portion of this sterile medium, ina flask, was inoculated with mycelial scrapings from an agar slant ofActinomadura madurae subspecies simaoensis NRRL 15734. The medium wasthen agitated vigorously on a rotary shaker for 48-72 hours at 28° C.providing primary inoculum. This primary inoculum was then used toinoculate 10 liters of the above sterile medium, which was then grown at28° C. for 48 hours, providing secondary inoculum. This secondaryinoculum was then used to inoculate 250 liters of the above sterilemedium, in a tank, which was grown for 48 hours at 28° C. with a flow ofsterile air of 200 liters per minute, providing tertiary inoculum.

EXAMPLE 2 Fermentation

A fermentation medium of the following formulation was prepared.

    ______________________________________                                        Sucrose          3.0%                                                         Soy flour        1.5%                                                         Corn steep liquor                                                                              0.5%                                                         Calcium carbonate                                                                              0.5%                                                         Water qs         100%                                                         ______________________________________                                    

This medium was sterilized and then inoculated with 125 liters oftertiary inoculum, prepared as described in Example 1, per 3000 litersof the above sterile fermentation medium. The fermentation was conductedat 28° C. with a sterile air flow of 6.6 liters per liter of mash,agitation by an impeller operated at 110 rpm and the addition ofsilicone defoamer agent for 137 hours, at which time the mash washarvested.

EXAMPLE 3 Isolation of LL-D42067β

A total of 4500 liters of harvest mash combined from two fermentationsconducted essentially as described in Example 2 was combined with 1% ofits volume of diatomaceous earth, mixed for one hour and then the pH wasadjusted to 3.0±0.3 with concentrated hydrochloric acid. One half themash volume of ethyl acetate was added and this mixture was stirred for3 hours. Diatomaceous earth equal to 5% of the mash volume was added andthe mixture was filtered. The ethyl acetate phase of the filtrate wasseparated, washed with 5% aqueous sodium bicarbonate and thenconcentrated to a syrup. This material was partitioned betweenheptane:methanol (2:1).

The 4.5 liters of methanol phase was concentrated to a residue which wastriturated with hexane. The hexane was decanted and the residueconcentrated to dryness. This material was purified by preparativereverse phase HPLC with the following conditions:

    ______________________________________                                        500/C.sub.18 Cartridge (aPak ®                                                        380 g silica-based octadecyl (C.sub.18) bonded                                phase packing material (dimensions: 5.7                                       cm × 30 cm), registered trademark of Wa-                                ters Associates, Inc., Milford, Massachu-                                     setts)                                                            Mobile Phase 1:                                                                           Acetonitrile:water:acetic acid                                                (8,000:12,000:10).                                                2:          Acetonitrile:water:acetic acid                                                (3,000:6,000:5).                                                  Flow Rate:  50 ml/minute.                                                     Fractionation:                                                                            200 ml/fraction.                                                  Sample Load:                                                                              2-3 g per 30 ml injection.                                        ______________________________________                                    

Using mobile phase 1, LL-D42067β was found in fractions 7-10. Thesefractions were combined and evaporated in vacuo to remove the bulk ofacetonitrile. The resulting aqueous suspension was treated with an equalvolume of ethyl acetate. The ethyl acetate phase was separated andsequentially washed with equal volumes of 5% aqueous sodium bicarbonate,0.1N hydrochloric acid and twice with water. The organic phase was driedover anhydrous sodium sulfate and evaporated to a solid.

This solid was rechromatographed using mobile phase 2. In this mobilephase LL-D42067β was found in fractions 15-21 which were combined andtreated as described above, giving pure LL-D42067β as a yellow solid.

We claim:
 1. A biologically pure culture of the microorganismActinomadura madurae subspecies simaoensis, NRRL 15734, said culturebeing capable of producing antibiotic LL-D42067β in recoverablequantities upon fermentation in an aqueous nutrient medium containingassimilable sources of carbon, nitrogen and inorganic anion and cationsalts.
 2. The biologically pure culture of the microorganismActinomadura madurae subspecies simaoensis according to claim 1, whereinsaid microorganism has spontaneously mutated, such that themicroorganism is genetically altered but still retains the ability tosynthesize antibiotic LL-D42067β.
 3. The biologically pure culture ofthe microorganism Actinomadura madurae subspecies simaoensis accordingto claim 1, wherein said microorganism has been subjected to mutagenicmeans such that the microorganism is genetically altered but stillretains the ability to synthesize antibiotic LL-D42067β.